Changes in gene transcription are important in the progression of cancer, in most other human diseases, and in the aging process, as well as in the development of multicellular organisms at all stages. A detailed understanding of how such changes are regulated is the basis of both diagnostic tools and intervention strategies. Further advancement holds the promise of novel approaches, and of increased effectiveness of current approaches. Importantly, many questions remain about the fundamental processes involved. Sequence-specific DNA binding proteins and the cofactors that they recruit to the DNA are among the most important regulators of transcription. One of the largest such families of proteins share the DNA binding homeodomain motif. Tools available in Drosophila make it possible to study mechanisms of action and interaction in detail in a true in vivo context. This proposal is to study mechanisms of action of several members of this family, to address basic questions that remain about how recognition of specific DNA target sites is accomplished, and the consequences of that recognition for the recruitment of cofactors to repress transcription, for chromatin changes that mediate regulation, and for the developmental pathways that are being regulated. Using a combination of in vivo and in vitro approaches, these studies will provide a clearer understanding of how combinations of proteins recognize appropriate target sites in vivo, and the consequences of that recognition for the regulation of downstream genes and pathways. The Specific Aims of the project are: 1) To determine the mechanisms that generate target gene specificity for the homeodomain protein Engrailed. Investigate the mechanisms that lead from DNA binding to repression of the direct target gene sloppy paired. 2) To analyze the functions of an En-interacting protein that contains a zinc-finger DNA binding domain, and to test whether this partner contributes to target gene specificity, or to activation/repression function on specific target sites in vivo. 3) To identify and analyze functional binding sites for the homeodomain-containing represser Even- skipped in the target gene sloppy paired. Determine the mechanisms responsible for the sequential repression during development of sloppy paired by first Even-skipped and then Engrailed.